Adjuvanted vaccine which is substantially free of non-host albumin

ABSTRACT

Disclosed herein is a serum-based adjuvanted vaccine which is substantially free of non-host albumin and the use thereof in reducing or preventing post-vaccination systemic reactions.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to serum-based vaccines that aresubstantially free of non-host albumin and processes for preparing andusing the same. More specifically, the present invention relates to theinventive concept of vaccines that prevent or substantially reducepost-vaccination adverse systemic reactions associated with adjuvantedvaccine regimens.

[0003] 2. Brief Description of the Prior Art

[0004] It is known in the art that vaccination of animals with vaccineregimens involving the use of adjuvants can cause adverse systemicreactions. The vaccine regimen can comprise administration ofinactivated vaccine containing an adjuvant. Alternately, the vaccineregimen can comprise administration of a modified live vaccine and aninactivated vaccine containing an adjuvant. Illustratively, most felinevaccine regimens comprise administration of a vaccine containing amodified live organism concomitantly with a vaccine containing aninactivated organism and an adjuvant. Associated with these vaccinationregimens are adverse systemic post vaccination reactions. For instance,the use of feline leukemia vaccines (FeLV) can cause post-vaccinationreactions including excess salivation, vomiting and diarrhea. See themonograph on FEL-O-VAX Lv-K vaccine in the Compendium of VeterinaryProducts, page 486, Third Edition, 1995-1996. The adverse systemicreactions include anaphylaxis, hypersensitivity and atypical reactionssuch as vomiting and diarrhea.

[0005] Contrary to the present inventive concept, the prior art hasattributed the above named systemic reactions to the presence ofadjuvants, endotoxins, cellular debris residue, high concentration ofmodified live viruses or high antigenic mass. Dodds, Vaccine Safety andEfficacy Revisited: Autoimmune and Allergic Diseases on the Rise, Vet.Forum, pp 68-71, May, 1993 noted an increase in post-vaccinationautoimmune and allergic diseases. Dodds has postulated that the increaseis due to the immunological burden on susceptible animals exposed to acombination vaccine containing modified live organisms and adjuvanted,killed bacterins administered at the same time (as the diluent). Doddsalso postulated that the immunological burden is produced by the effectof the modified live organisms.

[0006] The search for safe and effective vaccines has been limited bythe paucity of information regarding the source of the problem ofpost-vaccination reactions. There is no indication in the literature orotherwise that teaches that these systemic reactions could be caused byan interaction of non-host albumin with an adjuvant. Indicating thecontrary is the prevalent use of non-host albumin in the presence ofadjuvants. Dogs receive adjuvanted rabies vaccine at the same time thatthey receive modified live combination vaccines containing non-hostalbumin. Cats receive adjuvanted FeLV vaccine in a vaccine regimencomprising the concomitant administration of a modified live vaccinecontaining non-host albumin. Also, combinations of albumin and adjuvantsare commonly used in the art to evaluate the effectiveness of adjuvants.Albumin, generally in the form of Bovine Serum Albumin (BSA), isformulated with various adjuvants and each formulation is injected intonon-bovine animals. The animals are bled at some later date and theirsera are measured for antibody responses to BSA. The animals showing thebest antibody responses are considered to have received the mosteffective adjuvants. Prince et al, U.S. Pat. No. 4,164,565 discloses theuse of non-host albumin as a stabilizer in vaccines. Wiedmeier et al.,Pediatric Research, Vol.3, page 262-267, September, 1987 disclosesreactivity in mice produced by immunization with Bordetelia pertussiscombined with Bovine albumin. Notably, Wiedmeier et al teaches that thecause of reactivity is the pertussis toxin in combination with albumin.

[0007] To help reduce the systemic reactions, one can purify vaccines toremove components thereof which presumably cause the systemic reactions.Animal vaccine preparations are typically purified by conventionalmethods such as filtration, diafiltration or centrifugation to removecomponents such as cells and cellular debris. Other methods ofpurification that yield highly purified antigens are seldom employedbecause they are cost prohibitive in the preparation of animal vaccines.Illustrative of the other methods of purification is columnchromatography, including ion exchange chromatography, molecular sievechromatography and hydrophobic interaction chromatography. Moreover,highly purified antigens are difficult to adjuvant with the commonlyused adjuvants because they are not effective enough to stimulate aprotective response with purified antigens. At any rate, thesepurification methods were not effective for removing non-host albuminfrom vaccines or precursors thereof.

[0008] The art has not attributed the cause of systemic reactions to thepresence of adjuvants and non-host albumin. Certainly, the art has notattributed the cause of systemic reactions to the presence of non-hostalbumin in the vaccine regimen involving the use of adjuvants.

[0009] By the present invention, it has been realized that the presenceof non-host albumin in an adjuvanted vaccine or vaccine regimen cancause systemic reactions. By the present invention, there is provided anovel serum-based adjuvanted vaccine or vaccine regimen that issubstantially free of non-host albumin and a method of preparing thesame.

SUMMARY OF THE INVENTION

[0010] In accordance with the foregoing, the present inventionencompasses a serum-based vaccine comprising an immunogenicallyeffective amount of an antigen and an adjuvant wherein said vaccine issubstantially free of non-host albumin. The term “serum-based” is usedherein to denote that the vaccines of the invention or their precursorsemploy serum including non-host serum. Typically, the serum is employedin growth media to enhance growth of organisms that are employed in thepreparation of the vaccine. By the term “precursor of the vaccine” ismeant vaccine components, particularly antigen, proteins other thanantigen, whole organisms and harvest material. By the term“immunogenically effective amount” is meant that the antigen contains aprotective component in a concentration that is sufficient to protectanimals from a target disease when an adjuvanted vaccine containing theantigen is administered to animals. By the term “antigen” is meant abiological material (natural, recombinant or synthetic) that stimulatesa protective immune response in animals. By the term “adjuvantedvaccine” is meant a vaccine containing an adjuvant, or a plurality ofvaccines administered as a part of a vaccine regimen wherein at leastone of the vaccines contains an adjuvant. By the term non-host albuminis meant albumin from the serum of an animal species other than theanimal species being vaccinated. Albumin is a simple protein found inserum and has a molecular weight of about 66,000 daltons. A vaccinewhich is substantially free of non-host albumin contains less than 1.0mg/mL of non-host albumin.

[0011] Also encompassed by the invention is a method of preparing theserum-based vaccine that is substantially free of non-host albumincomprising removing non-host albumin from the vaccine or a precursorthereof. An alternate method of preparing the serum-based vaccine thatis substantially free of non-host albumin comprises providing a hostserum containing host albumin in the preparation of the vaccine.

[0012] Further encompassed by the invention is a vaccine which isprepared by adding host serum or albumin to the vaccine antigen afterharvesting or purifying the antigen from a culture of an organism fromwhich the antigen is derived, but prior to adjuvanting the antigen.Additionally, the host serum or albumin can be added to the antigenafter harvesting but prior to lyophilizing the antigen if the antigen isa modified live organism. When host serum or host albumin is used inthis manner, it acts as a stabilizer. The term “stabilizer” means anyadditive that is added to a vaccine to prevent degradation of theantigen and the consequential loss of immunogenicity of the vaccine.

[0013] In a presently preferred embodiment of the invention, the methodof preparing a serum-based vaccine containing an immunogenicallyeffective amount of an antigen and an adjuvant wherein said vaccine issubstantially free of non-host albumin comprises:

[0014] (a) growing an organism which produces the antigen in a culturecontaining non-host albumin;

[0015] (b) harvesting the culture;

[0016] (c) clarifying the harvest;

[0017] (d) separating the antigen and non-host albumin from theclarified harvest;

[0018] (e) separating the non-host albumin from the antigen;

[0019] (f) collecting the antigen; and

[0020] (g) formulating the antigen with an adjuvant.

[0021] In an additional preferred embodiment of the invention, themethod of preparing a serum-based vaccine containing an immunogenicallyeffective amount of an antigen and an adjuvant wherein said vaccine issubstantially free of non-host albumin comprises:

[0022] (a) growing an organism which produces the antigen in a culturecontaining non-host albumin;

[0023] (b) harvesting the culture;

[0024] (c) clarifying the harvest;

[0025] (d) separating the antigen from the non-host albumin by passingthe clarified harvest through a column with a matrix which selectivelybinds the antigen;

[0026] (e) washing the column matrix to remove excess non-host albumin;

[0027] (f) discarding the wash solution;

[0028] (g) washing the column matrix with a solution which elutes theantigen from the column matrix;

[0029] (h) collecting the antigen; and

[0030] (i) formulating the antigen with an adjuvant.

[0031] In another preferred embodiment of the invention, the method ofpreparing a serum-based vaccine containing an immunogenically effectiveamount of an antigen and an adjuvant wherein said vaccine issubstantially free of non-host albumin comprises:

[0032] (a) growing an organism which produces the antigen in a culturecontaining non-host albumin;

[0033] (b) harvesting the culture;

[0034] (c) clarifying the harvest;

[0035] (d) separating the antigen from the non-host albumin by passingthe clarified harvest through a column with a matrix which selectivelybinds the non-host albumin;

[0036] (e) collecting the antigen; and

[0037] (f) formulating the antigen with an adjuvant.

[0038] In still another preferred embodiment of the invention, themethod of preparation of a serum-based vaccine containing animmunogenically effective amount of an antigen and an adjuvant whereinsaid vaccine is substantially free of non-host albumin comprises:

[0039] (a) growing an organism which produces the antigen in a culturecontaining host albumin;

[0040] (b) harvesting the culture;

[0041] (c) clarifying the harvest, if necessary; and

[0042] (d) formulating the harvest with an adjuvant.

[0043] Further encompassed by the invention is a method of eliminatingadverse vaccine reactions in animals comprising administering to saidanimals a vaccine regimen which is substantially free of non-hostalbumin.

[0044] The method for eliminating adverse reactions in animals comprisesadministering to said animals an adjuvanted vaccine or an adjuvantedvaccine regimen which is substantially free of non-host albumin.

[0045] Also encompassed by the invention is a process for stabilizing anantigen comprising adding host serum or host albumin to said antigenprior to adjuvanting the antigen. Such a process for stabilizing anantigen can also comprise adding host serum or host albumin to saidantigen prior to lyophilizing the antigen.

[0046] The vaccines of the invention are applicable for use inpreventing or treating diseases of all species of animals. They areparticularly suitable for use in preventing or treating diseases ofcompanion animals such as cats, dogs and horses which are particularlysensitive to adjuvanted vaccine regimens comprising non-host albumin. Inparticular, the vaccines of the invention are suitable for use inpreventing feline leukemia (FeLV) and rabies because they are free ofproblems that typically attend such vaccines FeLV vaccines are notoriousfor causing adverse reactions such as hypersalivation, vomiting,diarrhea and sometimes death. Often, these reactions occur withinminutes of administration of the vaccine.

[0047] Surprisingly, it has been found that animals to which thevaccines of the invention have been administered have virtually noadverse systemic reactions. The discovery that non-host albumin in avaccine containing an adjuvant or administered in a vaccine regimen witha vaccine containing an adjuvant can cause systemic reactions is thus apart of the invention. This and other aspects of the invention aredescribed more fully hereunder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 is a graph presenting a comparison of the reactivity of avaccine containing an adjuvant combined with non-host albumin with thelack of reactivity of a vaccine containing an adjuvant in combinationwith host albumin.

[0049]FIG. 2 is a photograph of a SDS-PAGE gel comparing 9 differentfeline vaccines wherein the non-host albumin content is shown.

DETAILED DESCRIPTION OF THE INVENTION

[0050] As set forth above, the present invention encompasses aserum-based vaccine comprising an immunogenically effective amount of anantigen and an adjuvant wherein the vaccine is substantially free ofnon-host albumin and methods of making and using the same. It alsoencompasses a vaccine regimen wherein at least one vaccine in theregimen contains an adjuvant and at least one vaccine in the regimencontains non-host albumin. In addition, it encompasses a process forstabilizing an antigen comprising adding host serum or host albumin tosaid antigen prior to adjuvanting the antigen. Such a process forstabilizing an antigen can also comprise adding host serum or hostalbumin to said antigen prior to lyophilizing the antigen.

[0051] Non-host albumin is derived from non-host serum that is typicallyused in growing organisms from which the antigens are derived. Typicalexamples of non-host serum (containing non-host albumin) can be selectedfrom the group consisting of bovine serum, fetal bovine serum, equineserum, fetal equine serum, sheep serum and goat serum. On the otherhand, if equine albumin is present in an equine vaccine, the vaccine isconsidered to contain host albumin.

[0052] The antigen is obtained from an organism selected from the groupconsisting of bacteria, virus, parasite, rickettsia and protozoa.Examples of the bacteria can be selected from the group consisting ofBordetella spp., Streptococcus spp., Staphylococcus spp., Clostridiumspp., Leptospira spp., Escherichia spp., Salmonella spp., Pasteurellaspp., Mycobacteria spp., Mycoplasma spp., Moraxella spp., Haemophilusspp., Borrelia spp., Fusobacteria spp., Bacteriodes spp. and Rhodococcusspp. Examples of the viruses can be selected from the group consistingof herpes viruses, parainfluenza viruses, reoviruses, rotaviruses,morbilliviruses, retroviruses, coronaviruses, adenoviruses, togaviruses,parvoviruses, parapox viruses, paramyxoviruses, cytomegaloviruses,arboviruses and hantaviruses. More specifically, such viruses wouldinclude but not be limited to feline leukemia virus, felinerhinotracheitis, feline calicivirus, feline panleukopenia virus, felineimmunodeficiency virus, feline infectious peritonitis virus, caninehepatitis, canine adenovirus type 2, canine parvovirus, rabies virus,canine parainfluenza virus, canine coronavirus, equine herpes viruses,equine influenza viruses and equine encephalomyelitis viruses. Examplesof parasites and protozoa can be selected from the group consisting ofNeospora spp., Toxoplasma spp., Dirofilaria spp., Cryptosporidium spp.,Giardia spp., Babesia spp. and Coccidia spp.. An example of rickettsiacan be selected from the group consisting of Chlamydia spp., PotomacHorse Fever, Ehrlichia canis, and other Ehrlichia spp..

[0053] The antigens can be obtained from a member selected from thegroup consisting of: a whole culture of an organism such as a wholeculture harvest, a partially purified whole culture harvest, a purifiedsubunit extracted from harvest, a subunit obtained via recombinanttechnology and expressed in the homologous or a heterologous organism, adeletion mutant of the whole organism (conventional or rDNA gene-deletedmutants), peptides, naked DNA, chemically synthesized antigens, reversetranscribed naked cDNA or combinations thereof.

[0054] Generally, the antigen can be produced by art-known techniques ofculturing and harvesting organisms, concentrating and/or conventionallypurifying antigens of such organisms. For example, the antigen can beproduced by: growing the selected organism in a culture having growthmedium containing a non-host serum (serum-based culture). Morespecifically, the organism can be grown in a tissue culture preparedfrom mammalian or plant cells wherein non-host serum is added to themedium to enhance the growth of the organism. The organism can also begrown in fermentation media wherein the organism grows without tissueculture but has added thereto a growth medium containing a non-hostserum. Typically, the non-host serum can be selected from the groupconsisting of fetal bovine serum, bovine serum, calf serum, fetal equineserum, horse serum, goat serum, lamb serum and sheep serum. At thecompletion of growth, the culture is harvested and, if necessary,conventionally purified by, say, filtration and/or ultrafiltration toremove cells, cellular debris and extraneous contaminants. However,these techniques do not remove the non-host albumin. At this point, theculture harvest still contains non-host albumin and would not beacceptable if combined with adjuvant and/or administered in a regimenwith an adjuvanted vaccine. Therefore, the resulting culture harvest isfurther purified in accordance with this invention to remove thenon-host albumin prior to its formulation into an adjuvanted vaccine.

[0055] In accordance with the invention, the non-host albumin can beremoved by a process of purifying the vaccine or a precursor of thevaccine in such a manner as would remove the non-host albumin. Theprocess of purifying the precursor of the vaccine can be done by achromatography technique selected from the group consisting of Perfusionchromatography™ (PerSeptive Biosystems), ion exchange chromatography,molecular sieve chromatography, hydrophobic interaction chromatography,affinity chromatography and combinations thereof. Preferably, theprocess of purification is by Perfusion chromatography™ usinghydrophobic interaction chromatography matrices or a combination ofhydrophobic interaction chromatography and ion exchange chromatography.The following is an illustrative but non-limiting description of thehydrophobic interaction chromatography with a Perfusion Chromatography™matrix utilizing POROS media (PerSeptive Biosystems).

[0056] Perfusion Chromatography™ is carried out using a matrix (POROSmedia) having large channeled pores which carry molecules swiftly intothe interior of each bead by convective flow as well as diffusive poresthat branch off the channeled pores providing a large internal surfacearea for binding. This pore combination provides high capacity, highresolution and high speed purification. Hydrophobic interactionchromatography involves the use of polar groups on an uncharged matrixto interact with polar residues (e.g. phenylalanine) on proteins,causing retardation and separation of proteins based on their relativehydrophobicities. The use of the POROS media matrix allows much greaterflow rates at higher pressures so that the purification time is reduced,thus reducing the cost and allowing chromatography to be cost effectivefor veterinary products.

[0057] Hydrophobic interaction chromatography is performed by adding ahigh ionic strength buffer to fluids of the culture harvest containingthe non-host albumin before adding such fluids to the hydrophobiccolumn. The column is washed several times with a high ionic strengthbuffer such as 20 millimolar (Mm) sodium phosphate/650 Mm sodium sulfatebefore addition of the high ionic strength buffered fluids of theculture harvest containing the non-host albumin (column feed material).Multiple column volumes of column feed material are run through thecolumn. The column matrix binds both the non-host albumin and theantigen (contained within the buffered fluids of the culture harvest).To elute non-host albumin from the column, the column is washed multipletimes with a high ionic strength buffer such as 20 Mm sodiumphosphate/650 Mm sodium sulfate or until the optical density reading ata wavelength of 280 nanometers (nm) of the eluate is less than 0.03. Theantigen (purified) is eluted from the column by washing the columnmatrix with multiple volumes of a low ionic strength solution which canbe sterile water. The purified antigen is collected in a separatecollecting vessel when the optical density of the eluate increases above0.15. Collection of the eluate ceases when the optical density of theeluate drops below 0.10.

[0058] Another method for removal of non-host albumin according to thisinvention encompasses use of affinity chromatography for binding ofeither the antigen or the non-host albumin. For instance, the antigencan be produced by art-known techniques of culturing and harvestingorganisms and clarifying, concentrating and/or conventionally purifyingantigens of such organisms as described previously. For removal of thenon-host albumin the clarified harvest can be added to a columncontaining a matrix which binds either the antigen or which binds thenon-host albumin. Such a matrix could be a lectin such as CibaCron™ Blue(Pharmacia) or Mimetic Blue (Affinity Chromatography Ltd.), both ofwhich bind non-host albumin, or a matrix which contains a polyclonal ormonoclonal antibody specific for the antigen or non-host albumin,whichever is to be bound to the matrix. The clarified harvest becomesthe column feed material and is added to the column. If the columncontains a matrix such as a lectin, a polyclonal antibody or amonoclonal antibody specific for non-host albumin, the non-host albuminis bound to the column and the antigen passes through the column and iscollected. The collected antigen is then formulated with adjuvant toprepare a vaccine. If the column contains a matrix such as a polyclonalantibody or monoclonal antibody specific for antigen, the clarifiedharvest material is added to the column and the antigen is bound to thematrix. The non-host albumin passes through the column and is discarded.Excess non-host albumin is removed from the column matrix by washingwith a buffer which does not remove the antigen. Then the matrix iswashed with a solution which elutes the antigen from the column matrix.Such washing and elution buffers can be based on pH, ionic strength orpolarity differences of the antigen to be eluted. The antigen is thencollected and formulated with an adjuvant to produce the vaccine. If alectin is used to bind non-host albumin, the antigen which is collectedwill have to be further purified through a second lectin column or byusing another type of chromatography to remove all of the non-hostalbumin.

[0059] If one has a whole organism such as a virus or bacteria or a verylarge antigen, for instance, one with a molecular weight greater than100,000 daltons, molecular sieve chromatography can be used to separatethe antigen from the non-host albumin which has a molecular weight ofonly about 66,000 daltons. Molecular sieve chromatography separatesmolecules on the basis of molecular weight. The matrix is selected sothat low molecular weight molecules such as non-host albumin passthrough the column at a faster rate than large molecular weightmolecules such as large antigens. Using this technique, the organism isgrown in a culture containing non-host albumin and harvested, clarifiedand/or concentrated and purified by conventional techniques as describedpreviously. In order to separate the non-host albumin from, forinstance, a whole virus, the virus is grown in tissue culture, harvestedby collecting the fluids from the tissue culture and clarified to removethe cellular debris. This clarified harvest is the column feed materialand is added to the column. The first fluid to pass through the columnis collected and discarded since it contains the non-host albumin. Thevirus passes through the column slower and can be washed into acollection vessel using buffers which do not harm the virus. Virus whichhas been collected in this manner can be formulated with an adjuvant toprepare a vaccine.

[0060] An alternate method of preparing the serum-based vaccinecontaining an immunogenically effective amount of an antigen and anadjuvant wherein said vaccine is substantially free of non-host albumincomprises culturing the organism in host serum wherein there is nonon-host albumin. By this method, one grows the organism in tissueculture or fermentation media containing host serum instead of non-hostserum. Conventional harvesting, concentration and purification can beused if a pure product is desired. No further purification to removenon-host albumin is required because the preparation does not containnon-host albumin. By this method, the crude harvest material can also beused to formulate the vaccine. Using this method the harvest materialcan simply be combined with adjuvant to formulate the vaccine.

[0061] Following the purification and/or removal of the non-host albuminor growth of the organism in host serum, the antigen is inactivated andadjuvanted by conventional techniques. Generally stated, the antigen canbe inactivated by treating it with an inactivating agent which does notdenature the protective component of the antigen. Specifically, theantigen can be inactivated by treating it chemically, by irradiation, byheating or by freeze-thaw. Illustratively, one can employ chemicalinactivating agents selected from the group consisting of formalin,beta-propiolactone, detergents and binary ethyleneimine. Different onesof these chemical inactivating agents are preferred for differentorganisms.

[0062] The inactivated antigen can also be concentrated or pooled withother harvested antigen prior to adjuvanting. The amount ofconcentration would be such that the average amount of antigen orRelative Potency (RP) value meets or exceeds the minimum acceptablevalue for a vaccine. The inactivated antigen may be concentrated up to100 fold, if necessary, by ultrafiltration with a molecular weightcut-off which will suitably maintain the antigen and allow contaminantsto pass through and be discarded or by differential centrifugation.After inactivation, the antigen value must be above the acceptableminimum level or RP. Then it is stored at temperatures from −70° C. to+10° C. until it is mixed or microfluidized with an adjuvant.

[0063] The inactivated antigen is formulated or combined with anadjuvant. Adjuvants are chemicals or bacterial or virus-derivedcomponents added to vaccines to enhance the production of an immuneresponse by the animal receiving the vaccine. Adjuvants fall into thegeneral categories of polymers, block co-polymers, oils, oil-in-water,aluminum salts, and bacterial and viral extracts. Most adjuvantsfunction by producing an irritation at the site of injection causingleukocytes (immune cells) to infiltrate the area and/or by producing adepot effect (holding the antigens at the injection site for as long aspossible). Some of the newer adjuvants act as slow-release mechanisms,releasing antigens encapsulated by them at a relatively slow rate. Evennewer adjuvants directly affect the B-cells or T-cells of the immunesystem and are called immune stimulators, immune regulators, immunemodulators or immune enhancers. If an adjuvant causes extensiveinfiltration of leukocytes to the injection site, swelling andinjection-site reactions will occur. The immune response to adjuvantsmay also enhance the reactivity to contaminants such as endotoxins,thereby increasing the probability of systemic reactions such asanaphylaxis. Therefore, although adjuvants are necessary for stimulationof the immune response by inactivated vaccines, they can producedetrimental side effects. The adjuvant is selected from the groupconsisting of polymers, block co-polymers, oils, oil-in-water,water-in-oil, aluminum salts, immuno-modulators and combinationsthereof. Preferably, the adjuvant is a polymer or block co-polymer. Theadjuvant can be employed in an amount of from 0.01% to 50%. The amountof adjuvant is strictly correlated to the type of adjuvant used.However, it is important that the adjuvant be employed in an effectiveamount to immunogenically stimulate the inactivated antigens. When usedin such an amount, adjuvants can stimulate adverse reactions to non-hostalbumin.

[0064] After inactivating and adjuvanting the antigen, the potency orRelative Potency (RP) of the antigen can be adjusted to an appropriatelevel which meets or exceeds the minimum acceptable amount of antigen toproduce an immunogenically effective vaccine. The tests used for suchpotency or relative potency testing are described hereunder. Theantigen(s) can be formulated with other antigens. For example,inactivated and adjuvanted feline leukemia virus prepared in accordancewith the invention can be formulated with feline calicivirus, felinepanleukopenia virus, feline rhinotracheitis virus and feline chlamydia.Additionally, inactivated and adjuvanted rabies virus prepared inaccordance with the invention can be formulated with canine parvovirus,canine distemper virus, canine parainfluenza virus, canine adenovirustype 2 and various Leptospira spp. Also, inactivated and adjuvantedequine viruses and bacterial antigens can be prepared in accordance withthe invention. Some or all of these additional antigens may be preparedaccording to the present invention. Some of the additional antigens maybe modified live. However, the final combination vaccines will besubstantially free of non-host albumin if the combination vaccine orvaccine regimen wherein the combination vaccine is administered containsan adjuvant. The resulting adjuvanted vaccine that is substantially freeof non-host albumin is safe and effective and can be administered toanimals with essentially no post-vaccination, adverse systemicreactions.

[0065] As a measure of vaccine potency that equates to vaccineprotection in the host animal, each individual lot of antigen (crude orpurified) and serial of vaccine undergoes testing. The measurement mayinvolve vaccination of laboratory animals or host animals followed by achallenge of the animals, vaccination of laboratory animals or hostanimals followed by evaluation of a serological response or theperformance of an Enzyme Linked Immunosorbant Assay (ELISA) to measurethe amount of antigens in the vaccine. An Enzyme Linked ImmunosorbantAssay (ELISA) is preferable as it eliminates animal testing. In thelatter method, the antigen concentration in the test vaccine is measuredagainst the antigen content in a Reference Vaccine which has been provento be protective in the host animal. A test vaccine which measures 1.0as compared with the Reference Vaccine is considered to be potent and issaid to have a relative potency (RP) of 1.0. The RP can be measuredbefore or after the antigen has been harvested, purified, inactivated oradjuvanted. Before inactivation and adjuvanting, the RP must be above1.0 so that after inactivation and adjuvanting it does not fall below1.0.

[0066] The purified antigen in accordance with the invention, may beconcentrated or pooled with other purified harvested antigen such thatthe average amount of antigen meets or exceeds the minimum acceptablevalue for a harvest. The purified antigen may be concentrated up to 100fold, if necessary, by ultrafiltration with a molecular weight cut-offwhich will suitably maintain the antigen and allow contaminants to passthrough and be discarded or by differential centrifugation. It isimportant to note that even if very low levels of serum are used forgrowth enhancement, it is virtually impossible to remove its albumincontent from cultures of the organism or vaccines by conventionalpurification processes, especially if concentration is used. Forinstance, if antigen is concentrated 100 fold, a non-host albumin levelof 0.1% (1 mg/mL) in the antigen prior to concentration would beconcentrated to 10% or 100 mg/mL after concentration. Such a level wouldbe totally unacceptable in the final vaccine.

[0067] As would be realized from the foregoing, a distinct feature ofthe invention is the discovery of the source of the problem ofpost-vaccination adverse systemic reactions and the solutions for theproblem. Without being bound to any particular theory, it is believedthat the adverse post-vaccination systemic reactions result from thepresence of adjuvants and non-host albumin in vaccines or vaccineregimens. There is hereby discovered and disclosed a solution whichincludes removing non-host albumin from vaccines which contain anadjuvant or which are administered in vaccine regimens which containadjuvanted vaccines or using host serum for antigen preparation in placeof non-host serum and administering vaccines and vaccine regimens whichare substantially free of the non-host albumin.

[0068] These and other aspects of the invention are further illustratedby the following non-limiting examples. In the examples and throughoutthe specification, parts are by weight unless otherwise indicated.

EXAMPLES Example 1

[0069] In order to evaluate whether there was a difference in reactivityof equine vaccines prepared with non-host serum (fetal bovine serum asthe conventional approach) or equine vaccines prepared with host serum(fetal equine serum) two mock vaccines were prepared. One vaccinecontained adjuvanted media with 15% fetal bovine serum (non-host serumapproach) while the second vaccine contained 15% fetal equine serum(host serum). Twenty horses were used for this study. The adjuvant inthe two approaches was from the same lot of material and was a“Carbopol” based adjuvant. Ten horses each received a 2.0 mL dose of thefetal bovine serum-containing mock vaccine injected intramuscularly inthe neck and each of an additional ten horses received a 2.0 mL dose ofthe fetal equine serum-containing mock vaccine injected intramuscularlyin the neck. A booster injection of the respective vaccines wasadministered every 28 days over approximately 8 months. The horses wereobserved for reactions on days 1, 2, 3, 4, 7 and 14 following eachinjection. Just before the second injection, one of the horses receivingthe fetal bovine serum preparation died of contortion of the intestine.The remaining 9 horses received a booster injection and were observedafter booster doses of the fetal bovine serum-containing mock vaccine.The results of these observations are shown in FIG. 1.

[0070] Following administration of all injections of the fetal equineserum-containing mock vaccine, there were no systemic reactions (0 outof a possible 480 observations). Only 2 out of a possible 480 instancesof swelling of 4″ or greater in diameter were observed. The swellingoccurred in 2 consecutive observations of the same horse after receiving4 injections. Swelling of 1-3″ in a diameter was observed in 3 out of apossible 480 observations. Thirty one (31) reactions of any type wereobserved out of a possible 480 observations. All reactions occurred inonly 1 of the 10 horses (10%) through vaccination # 8 after which 2 ofthe 10 horses showed a local reaction. Comparatively, followingadministration of all injections of the fetal bovine serum mock vaccine,there was one possible systemic reaction (the death of horse #606).Severe swelling (larger than 4″ in a diameter) was observed in 22 out ofa possible 432 observations. Visible swelling (1-3″ in a diameter) wasobserved in 34 out of a possible 432 observations. One hundred andforty-six (146) reactions were noted out of a possible 432 observations.Eight of the remaining 9 horses (89%) showed reactivity by vaccination#8 with 5 of 9 horses reacting routinely after each vaccination. Thesedata indicate that in repeat injection with adjuvanted vaccines, thepresence of non-host serum (fetal bovine serum in equine vaccines)causes considerably more reaction than the presence of host serum (fetalequine serum).

Example 2A

[0071] CRFK cells (Crandell Feline Kidney) persistently infected withFeLV were grown to 95% confluency as follows. The cells were grown in850 cm2 roller bottles incubated with rotation at 37° C. Employed as thegrowth medium was Dulbecco's Minimal Essential Medium with high glucoselevels (DMEM-Hi) containing 10% fetal bovine serum and 30 ug/mlneomycin. After the cells reached confluency, the media was changed tomaintenance media (DMEM-Hi media containing 5% fetal bovine serum).After four days this media was decanted and viral fluids were harvested.Cells were re-fed with maintenance media and viral fluids were collectedevery three to four days for a total of seven harvests. Decanted viralfluids from each harvest were tested for sterility, aliquoted intosterile plastic containers and stored frozen at −70° C. Uponsatisfactory sterility testing, viral fluids were thawed at roomtemperature and pooled into a single sterile receiving vessel. Viralfluids were clarified through a 3 micron polypropylene filter to removecell debris and then concentrated 10-fold using a 30,000 daltonmolecular weight cut-off tangential flow ultrafiltration device. Fluidswere then washed in 50 Mm Na₂HPO₄ to a 9-fold final concentrationfactor. The pooled concentrate had a total protein content of 16.59mg/mL.

[0072] A cation exchange chromatography column was initially used topurify the virus and its subunits from the remainder of the fluids. Aone-liter, 14 cm×10 cm column was packed with Q Sepharose chromatographyresin (Pharmacia) and sanitized with two column volumes of 1 M NaOH. Inaddition, the column accessories such as pumps, tubing and fittings tothe column were sanitized with 1M NaOH. The column and all accessorieswere then rinsed with a 50 mM Na₂HPO₄ (pH 7.0) buffer until the effluentfrom the column was at pH 7.0. All buffers were 0.2 μm filter sterilizedbefore use. A BioPilot Chromatography system (Pharmacia) was thehardware used for this entire process.

[0073] A 1500 mL sample of the FeLV concentrated viral fluids (columnfeed material) was injected onto the column. The column was washed with9 column volumes of Buffer A (50 mM Na₂HPO₄) at 80 mL/min before elutionof the virus with a linear gradient of Buffer B (50 mM Na₂HPO₄,1 M NaCl)from 0% to 50% Buffer B within 10 column volumes. A final elution ofresidual virus was accomplished with 5 column volumes of 100% Buffer B.Fractions eluted from the column were collected and examined for totalprotein content. The total protein content was 3.12 mg/mL. Approximatelyhalf of this would be non-host albumin. Fractions containing the viruswere then pooled and rechromatographed over a hydrophobic interactioncolumn to remove the remainder of the non-host albumin content.

[0074] Ammonium sulfate was added to the eluted virus fractions from theQ Sepharose column (which still contained >1 mg/mL of non-host albumin)to achieve a final concentration of 0.5 M. This virus fraction columnfeed material was loaded onto a 1 liter phenyl sepharose lowsubstitution hydrophobic interaction column that had been previouslyequilibrated with 50 mM Na₂HPO₄. The column was washed with acombination of 50 mM Na₂HPO₄ and 0.5 M (NH₄)₂SO₄ for 5 column volumes.The virus was then eluted from the column with a 50 mM Na₂HPO₄ buffer.Virus fractions eluted from the column were tested for sterility, totalprotein and non-host albumin content. All virus fractions were sterile,the total protein content was between 0.9 and 1.2 mg/mL and the non-hostalbumin content was below 0.5 mg/mL. The viral TABLE 1 FELINE LEUKEMIAVIRUS VACCINATION/CHALLENGE STUDY - DETECTION OF PERSISTENT VIREMIA BYMEASUREMENT OF p27 Test CAT Wk Wk Wk Grp. NO. D-15 D-1 D 34 Wk 3 Wk 4 Wk5 Wk 6 Wk 7 Wk 8 Wk 9 10 11 12 FLV VL2 − − − − − − − − − − − − − 009 VM3− − − − − − − − − − − − − VQ2 − − − − − − − − − − − − − VV4 − − − − − −− − − − − − − VX2 − − − + + + + + + + + + + WC1 − − − − − − − − − − − −− WE3 − − − + + + + + + + + + + WF6 − − − − − − − − − − − − − WH5 − − −− − − − − − − − − − WK3 − − − + + + + + + + − − − FLV VL5 − − − − − − −− − − − − − 011 VN1 − − − − − − − − − − − − − VW3 − − − − − − − − − − −− − VY4 − − − − − − − − − − − − − WD2 − − − − − − − − − − − − − WF1 − −− − − − − − − − − − − WG3 − − − − − − − − − − − − − WJ4 − − − − − − − −− − − − − WL5 − − − − − − − − − − − − − VT5 − − − − − − − − − − − − −Cont VL1 − − − − − − − − − − − − − VM2 − − − + + + + + + + + + + VQ1 − −− − − − − − − − − − − VV3 − − − + + + + + + + + + + VX1 − −− + + + + + + + + + + WB − − − + + + + + + + + + + WE2 − −− + + + + + + + + + + WF3 − − − + + + + + + + + + + WH4 − −− + + + + + + + + + + WJ6 − − − + + + + + + + + + + WM − −− + + + + + + + + + +

[0075] fraction (fluids) was inactivated with 0.03% formalin andformulated into vaccines by combining with either 5% POLYGEN™ adjuvant(obtained from MVP Laboratories, Ralston, Nebraska), 0.25% glycerol/EDTAstabilizer and 30 ug/mL nystatin (FLV011) or 0.125% carbopol adjuvant,0.25% glycerol/EDTA stabilizer and 30 ug/mL nystatin (FLV009).

[0076] Ten to twelve week old cats were immunized with a one mL dose ofvaccine subcutaneously. Three weeks later the cats were given a one mLbooster immunization. Cats were challenged ten days post boostervaccination with virulent feline leukemia virus. This challenge wasconducted as follows: 1) cats were immunosuppressed with 10 mg/kg bodyweight of methylprednisolone acetate intramuscularly for two successivedays; and 2) cats were challenged with approximately 1.5×10⁶ focusforming unit (FFU) of virulent feline leukemia virus intranasally oneach day of immunosuppression. Cats were checked at day 15 and day 1prior to challenge exposure to make sure that they were not alreadyinfected with FeLV or were not carriers. Beginning three weeks afterchallenge, blood was collected from cats for nine successive weeks andexamined for “p27e” antigen by an indirect immunofluorescence assay. Allresults of the vaccinates and control cats are presented in Table 1. Apositive test result for a cat was defined as three consecutive weeks ofviremia or five weeks of viremia during the twelve week period. Theresults indicate that 100% of the cats vaccinated with “FLV011” vaccineand seventy percent of the cats vaccinated with “FLV009” vaccine wereprotected from challenge, whereas, 81 percent of control cats wereinfected by the challenge dose. This is equal to or better than theprotection provided by conventionally-produced but reactive commercialFeLV vaccines which protect from 15 to 80 percent of the vaccinated catsin a similarly intense challenge. This FLV009 vaccine serial became theStandard Reference for future ELISA assays and, by definition, containsan RP of 1.0.

Example 2B

[0077] The vaccine of EXAMPLE 2A was evaluated for safety by 21practicing veterinarians in clinical field trials conducted in fivestates. A total of 913 doses of vaccine were administered to 850 catsbetween 8 weeks and 15 years of age. The veterinarians were requested tospecifically note any systemic reactions and record the circumstancessurrounding such incidences should they occur. Only one systemicreaction was noted. This reaction occurred in a cat which received aconcomitant modified live feline combination vaccine which containednon-host albumin. Therefore, it is concluded that the FeLV vaccine wassafe and that systemic vaccine reactivity can be eliminated byadministering vaccines which do not contain a combination of non-hostalbumin and an adjuvant whether administered in the same vaccine orwhether administered in a concomitant vaccine as part of a vaccinationregimen.

Example 3

[0078] CRFK cells persistently infected with FeLV were grown to 95%confluency in DMEM-Hi containing 10% fetal bovine serum and 30 ug/mLneomycin using 850 cm² roller bottles incubated with rotation at 37° C.as in EXAMPLE 2A. After the cells reached confluency, the media waschanged to maintenance media (DMEM-Hi media containing 5% fetal bovineserum). After four days, this media was decanted and viral fluids wereharvested. Cells were refed with maintenance media and viral fluids werecollected every three to four days for a total of seven harvests.Decanted viral fluids from each harvest were tested for sterility. Allharvest fluids were found to be sterile. Viral fluids from each harvestwere aliquoted into sterile plastic containers and stored frozen at −70°C. Upon satisfactory sterility testing, viral fluids were thawed at roomtemperature and pooled into a single sterile receiving vessel. The totalprotein content of this pooled FeLV was 2.5 mg/mL. Viral fluids wereclarified through a 5 micron and a 1 micron polypropylene filter toremove cell debris.

[0079] Clarified viral fluids (harvest fluid column feed material) werepurified to remove non-host albumin by using a purification techniquecomprising Perfusion Chromatograph™ using a hydrophobic interactionchromatography matrix. The matrix used was obtained from PerSeptiveBiosystems and was their POROS PE 50 media. This technique uses polargroups on an uncharged matrix to interact with polar residues (e.g.phenylalanine) on proteins, causing retardation and separation ofproteins based on their relative hydrophobicities. This interaction wasenhanced by adding high ionic strength sodium sulfate/sodium phosphateto the viral fluids before adding them to the hydrophobic column. Thecolumn was washed with three column volumes of a buffer containing 20 mMsodium phosphate, 650 mM sodium sulfate before addition of harvest fluidcolumn feed material. The equivalent of five column volumes of harvestfluid column feed material (before dilution with sodium sulfate/sodiumphosphate) was then run through the column. To elute non-host albuminfrom the column, the column was washed with five column volumes of 20 mMsodium phosphate/650 mM sodium sulfate or until the optical densityreading of the eluate was <0.03 at a wavelength of 280 nm. Resultantpurified viral components were eluted from the column by washing thecolumn resin with five column volumes of sterile water. Purified viralfractions were collected in a separate collecting vessel when theoptical density (at 280 nm) of the eluate increased above 0.15 andcollection of the eluate ceased when the optical density of the eluatedropped below 0.10.

[0080] Purified viral fluids were tested quantitatively for totalprotein content and qualitatively by SDS-PAGE for non-host albumincontent. The total protein was 1.35 mg/mL and the non-host albumincontent was less than 0.5 mg/mL. To remove excess salts which areretained by the purified virus fluids, these fluids were diafilteredwith ten volumes of Dulbecco's phosphate buffered saline using a 30,000dalton molecular weight cut-off tangential flow ultrafiltration device.Fluids were concentrated at this time to achieve a sufficient finalconcentration of FeLV gp70 to batch vaccine. Viral fluids wereinactivated with 0.03% formalin for 72 hours at 4° C.

[0081] A feline leukemia vaccine was produced from purified, inactivatedviral fluids by addition of 0.125 mg/ml Carbopol, 0.25% glycerol/EDTAand 30 ug/ml nystatin to the inactivated viral fluids at a sufficientconcentration to be immunogenically effective when combined with theadjuvant. This vaccine was compared to the vaccine in EXAMPLE 2A usingan ELISA (Enzyme Linked immunosorbant Assay) to measure potency(immunogenic effectiveness). This ELISA measures the amount of antigeniccomponent in the FeLV vaccine as compared with a Standard Reference. TheStandard Reference is the vaccine described in EXAMPLES 2A and 2B (FLV009), which has been demonstrated to protect cats in avaccination/challenge study. A result of 1.00 in the ELISA indicatesthat the amount of FeLV protective antigen component in the vaccinebeing tested is equivalent to that of the vaccine of EXAMPLES 2A and 2Band will protect cats equally well. The vaccine of this exampledemonstrated a potency of 1.32. It contained a protein content of 1.1mg/mL with no detectable non-host albumin.

[0082] It is thus demonstrated that passage of FeLV harvest materialcontaining non-host albumin through a hydrophobic matrix alone canremove the albumin and be used to produce an immunogenically effectivevaccine.

Example 4

[0083] Six mock vaccines were formulated containing only 5% bovinefraction V albumin (non-host albumin) diluted in phosphate bufferedsaline and combined with different adjuvants. The vaccine formulationswere administered to fifty eight (58) cats of approximately 22-26 weeksof age that had previously received two doses of a combination killedrhinotracheitis virus-calicivirus-panleukopenia virus vaccine thatcontained serum proteins from tissue culture components. The cats hadalso been vaccinated with a purified feline leukemia vaccine containingno detectable non-host albumin. Cats were randomly assigned to annon-host albumin/adjuvant vaccine group and were immunized with a 1.0 mLdose of vaccine weekly for 3 successive weeks. Daily observations weremade for evaluation of reactivity. Results of these observations areshown in TABLE 2. Approximately six hours after the first weeklyvaccination, two cats showed clinical signs of systemic reactions. Thevaccinations given to these cats were of two different non-hostalbumin/adjuvant formulations. Clinical signs included weakness andincoordination, vocalization, blood-tinged frothy vomiting, cyanoticextremities, pale mucous membranes with delayed capillary refill time(3.5 seconds), hypersalivation and hyperpnea. Treatment withdexamethasone and subcutaneous fluids did little to relieve symptoms.These two cats were subsequently euthanized.

[0084] Approximately four hours after the second weekly injection, oneadditional cat demonstrated more moderate signs of a systemic reactionto vaccination. Clinical signs associated with this animal includedlethargy, red mucous membranes, cyanotic/reddened pinna and swolleneyes. This experiment proves that the unusual reactivity commonlyassociated with FeLV vaccines (vomiting and diarrhea) can be reproducedby a combination of non-host albumin with an adjuvant. The reaction ratewhich was demonstrated in this experiment appears low. However, in theclinical situation, such reactions are only seen in less than 1.0% ofcats. This experiment demonstrated a 2.0% reaction rate if all cats areincluded in the calculation. TABLE 2 DEMONSTRATION THAT NON-HOST ALBUMININ COMBINATION WITH ADJUVANTS PRODUCES SYSTEMIC REACTIONS IN ANIMALS NO.OF REACTIONS/ VACCINE AMT. OF NO. OF CATS NUMBER ADJUVANT ADJUVANT WK 1WK 2 WK 3 1 NONE N/A 0/8 0/8 0/8 2 ADJUVANT B 50%  0/12  1/12  0/12 3CARBOPOL 0.125  1/8* 0/7 0/7 mg/mL 4 CARBOPOL 0.25  0/6 0/6 0/6 mg/mL 5ALUMINUM 10% 0/8 0/8 0/8 HYDROXIDE 6 EMULSIGEN  5% 0/8 0/8 0/8 7 POLYGEN 5%  1/8* 0/7 0/7

Example 5

[0085] Combination five-way feline vaccines prepared from modified liveor inactivated feline rhinotracheitis, feline calicivirus, felinepanleukopenia, feline chlamydia and feline leukemia virus are known inthe art and are also associated with the reactivity describedpreviously. A combination inactivated feline rhinotracheitis, felinecalicivirus, feline panleukopenia, feline chlamydia and feline leukemiavaccine was prepared according to this invention. The felinerhinotracheitis virus, feline calicivirus, feline panleukopenia virusand feline chlamydia virus protective antigen components were grownindividually in tissue culture without the use of serum or albumin. Theprotective antigen components were harvested and all contained celldebris. The individual harvest fluids were clarified through 3 μmfilters, inactivated individually with 0.1 M binary ethyleneimine andadjuvanted individually with 5% Polygen™. The feline leukemia used forthis combination was that prepared in EXAMPLE 3. Individuallyinactivated and adjuvanted components were combined in proportionsadequate to produce a 1.0 mL dose volume. The five protective antigencomponents were all demonstrated to be immunogenically effective bytesting in the specific ELISA tests as described in EXAMPLE 3. Thefeline rhinotracheitis component had an RP of 1.12 as compared to avaccine with a value of 1.0 which protected 100% of cats in a severevaccination/challenge test. The feline calicivirus componentdemonstrated an RP of 1.69 as compared to a vaccine with a value of 1.0which protected 100% of cats in a severe vaccination/challenge test. Thefeline panleukopenia virus showed an RP of 1.26 as compared to a vaccinewith a value of 1.0 which protected 100% of cats in a severevaccination/challenge test. The chlamydia component was tested for itsprotective capability in a cat vaccination/challenge test. It protected100% of the vaccinated cats.

[0086] The five-way feline combination of this invention was compared tocompetitor products which are known to cause reactivity of the kinddescribed using SDS-PAGE. FIG. 2 is a photograph of this gel whichdemonstrates the amounts of non-host albumin and other proteins in thevarious products. It should be noted that such electrophoretictechniques have been demonstrated to detect albumin at levels of 0.5mg/mL in vaccines. Gel lanes are numbered from left to right with #1being the farthest left lane and #10 being the farthest right lane. InFIG. 2, lanes 1, 2 and 4 are monovalent FeLV vaccines which were foundto be highly reactive in the field. They contain a significant amount(>1.0 mg/mL) of non-host albumin which is detectable by a band atapproximately 66,000 daltons. These vaccines caused systemic reactionsof the type previously described, as well as death in a significantnumber of animals when tested in field safety trials similar to thetrial described in EXAMPLE 2B. Lane 3 is a molecular weight marker whichcontains bands at 14.3, 20, 29, 34.8, 58.1 and 97 kilodaltons. Lane 5 isthe vaccine made according to EXAMPLE 2A which produced no systemicreactions when tested for safety in the field trial described in EXAMPLE2B. It contains a nondetectable level of non-host albumin. The bandwhich appears at 70 kilodaltons is indicative of the presence of thegp70 antigenic component. Lane 6 is 5% bovine serum albumin (BSA) whichserves as a non-host albumin control. Note that the gp70 band in lane 5is slightly higher than the midpoint of this non-host albumin band. Lane7 is a 5-way inactivated combination feline vaccine with the samecomponents as mentioned above which is marketed by Fort DodgeLaboratories under the name EEL-O-VAX^(R) Lv-K IV. As mentionedpreviously, the Compendium of Veterinary Products indicates thatsystemic reactions have been associated with this vaccine. Lane 8 is a5-way modified live/inactivated combination feline vaccine with the samecomponents as mentioned above which is marketed by Solvay under the nameEclipse^(R) 4+FeLV, This vaccine is also known to be reactive in cats.Lane 9 contains a 3-way modified live combination feline vaccinecontaining only feline rhinotracheitis, feline calicivirus and felinepanleukopenia which is marketed by Intervet under the name PROTEX™ -3.This vaccine does not contain FeLV so there should be no gp70 present.Therefore, the band at 66 kilodaltons is non-host albumin. This vaccine,when combined with an inactivated and adjuvanted FeLV vaccine in thesame vaccine regimen, caused systemic reactions in a study conducted incollaboration with the inventors. Lane 10 is a 3-way modified livecombination feline vaccine containing feline rhinotracheitis, felinecalicivirus and feline panleukopenia which contains no adjuvant and ismarketed by Solvay under the name Eclipse^(R) -3. It does not containFeLV so it should show no bands between 60 and 70 kilodaltons. However,there is a faint band at approximately 66 kilodaltons indicating thatthis vaccine contains non-host albumin. It would be expected that thisproduct, when used in a vaccine regimen with an adjuvanted vaccine wouldproduce systemic reactions. It is obvious from this Figure that the FeLV5-Way combination vaccine made according to this invention contains theleast amount of non-host albumin of any vaccine containing the fivecomponents. It is also obvious that all of the reactive vaccines containa marked band which represents non-host albumin at a concentration above0.5 mg/mL.

[0087] Although the invention has been described in detail in theforegoing for the purpose of illustration, it is to be understood thatsuch detail is solely for that purpose and that variations can be madetherein by those skilled in the art without departing from the spiritand scope of the invention except as it may be limited by the claims.

What is claimed is:
 1. A serum-based vaccine comprising animmunogenically effective amount of an antigen and an adjuvant whereinsaid vaccine is substantially free of non-host albumin.
 2. Theserum-based vaccine of claim 1 wherein the antigen is selected from thegroup consisting of a virus, a bacteria, a rickettsia, a parasite, aprotozoa, subunits therefrom and combinations thereof.
 3. Theserum-based vaccine of claim 2 wherein the virus antigen is selectedfrom the group consisting of retroviruses, herpes viruses, adenoviruses,paramyxoviruses, coronaviruses, morbilliviruses, hantaviruses,reoviruses, rotaviruses, togaviruses, parvoviruses, parapox viruses,cytomegaloviruses, arboviruses, and parainfluenza viruses subunitstherefrom and combinations thereof.
 4. The serum-based vaccine of claim2 wherein the bacterial antigen is selected from the group consisting ofClostridium spp., Streptococcus spp., Staphylococcus spp., Bordetellaspp., Pasteurella spp., Salmonella spp., Mycobacteria spp., Mycoplasmaspp., Leptospira spp., Borrelia spp., Fusobacteria spp., Bacteriodesspp., Rhodococcus spp., Escherichia spp., Salmonella spp., Moraxellaspp., Haemophilus spp., subunits therefrom and combinations thereof. 5.The serum-based vaccine of claim 2 wherein the rickettsial antigen isChlamydia spp., Ehrlichia spp. or subunits therefrom and combinationsthereof.
 6. The serum-based vaccine of claim 2 wherein the parasiteantigen is selected from the group consisting of Toxoplasma spp.,Dirofilaria spp., Cryptosporidium spp., Coccidia spp., Babesia spp.,Neospora spp., subunits therefrom and combinations thereof.
 7. Theserum-based vaccine of claim 1 wherein the adjuvant is selected from thegroup consisting of polymers, block co-polymers, oils, oil-in-water,aluminum salts and non-specific immunostimulants.
 8. The serum-basedvaccine of claim 1 wherein the adjuvant is present in an amount of from0.01 to 50%.
 9. The serum-based vaccine of claim 1 wherein the non-hostalbumin has been removed.
 10. The serum-based vaccine of claim 1 whereinsaid serum-based vaccine contains less than 1.0 mg/mL of non-hostalbumin.
 11. A method of preparing a serum-based vaccine containing animmunogenically effective amount of an antigen and an adjuvant which issubstantially free of non-host albumin comprising removing non-hostalbumin from the vaccine or a precursor of the vaccine.
 12. The methodof preparing a serum based vaccine as recited in claim 11 wherein theremoval of non-host albumin comprises purifying the vaccine or aprecursor of the vaccine to remove the non-host albumin.
 13. The methodof preparing a serum-based vaccine as recited in claim 12 wherein thepurification comprises column chromatography.
 14. The method ofpreparing a serum-based vaccine as recited in claim 13 wherein the typeof column chromatography is selected from the group consisting ofPerfusion, ion exchange, hydrophobic interaction, affinity, molecularsieve and combinations thereof.
 15. A method of preparing a serum-batedvaccine containing an immunogenically effective amount of an antigen andan adjuvant wherein said vaccine is substantially free of non-hostalbumin comprising: (a) growing an organism which produces the antigenin a culture containing non-host albumin; (b) harvesting the culture;(c) clarifying the harvest; (d) separating the antigen and non-hostalbumin from the clarified harvest; (e) separating the non-host albuminfrom the antigen; (f) collecting the antigen; and (g) formulating theantigen with an adjuvant.
 16. The method of claim 15 wherein the step(d) of separating the antigen and non-host albumin from the clarifiedharvest comprises passing the clarified harvest through a column havinga matrix which binds the non-host albumin and the antigen.
 17. Themethod of claim 15 wherein the step (e) of separating the non-hostalbumin from the antigen comprises selective elution of the antigen andnon-host albumin from the column matrix.
 18. A method of preparing aserum-based vaccine containing an immunogenically effective amount of anantigen and an adjuvant wherein said vaccine is substantially free ofnon-host albumin comprising: (a) growing an organism which produces theantigen in a culture containing non-host albumin; (b) harvesting theculture; (c) clarifying the harvest; (d) separating the antigen from thenon-host albumin by passing the clarified harvest through a column witha matrix which selectively binds the antigen; (e) washing the columnmatrix to remove excess non-host albumin; (f) discarding the washsolution; (g) washing the column matrix with a solution which elutes theantigen from the column matrix; (h) collecting the antigen; and (i)formulating the antigen with an adjuvant.
 19. A method of preparing aserum-based vaccine containing an immunogenically effective amount of anantigen and an adjuvant wherein said vaccine is substantially free ofnon-host albumin comprising: (a) growing an organism which produces theantigen in a culture containing non-host albumin; (b) harvesting theculture; (c) clarifying the harvest; (d) separating the antigen from thenon-host albumin by passing the clarified harvest through a column witha matrix which selectively binds the non-host albumin; (e) collectingthe antigen; and (f) formulating the antigen with an adjuvant.
 20. Amethod of preparing a serum-based vaccine containing an immunogenicallyeffective amount of an antigen and an adjuvant comprising: (a) growingan organism which produces the antigen in a culture containing non-hostalbumin; (b) harvesting the culture; (c) clarifying the harvest; (d)separating the antigen and non-host albumin from the clarified harvestusing Perfusion Chromatography and POROS PE 50 media; (e) washing thenon-host albumin from the POROS PE 50 media with a high ionic strengthbuffer; (f) eluting the antigen from the POROS PE 50 media using a lowionic strength solution; (g) collecting the antigen; and (h) formulatingthe antigen with an adjuvant.
 21. The serum-based vaccine of claim 1further comprising host albumin.
 22. A method of preparing theserum-based vaccine of claim 21 containing an immunogenically effectiveamount of an antigen and an adjuvant wherein said vaccine issubstantially free of non-host albumin comprising: (a) growing anorganism which produces the antigen in a culture containing hostalbumin; (b) harvesting the culture; (c) clarifying the harvest ifnecessary; (d) formulating the harvest with an adjuvant.
 23. A processfor reducing or preventing non-host albumin stimulated adverse vaccinereactions in animals comprising administering to said animals a vaccinewhich is substantially free of non-host albumin.
 24. A serum-basedvaccine comprising an immunogenically effective amount of an antigen, anadjuvant and a host serum or host albumin which is added after harvestor purification of the antigen but prior to adjuvanting the antigen. 25.A method of preparing a serum-based vaccine containing animmunogenically effective amount of an antigen and an adjuvant by addinga host serum or host albumin after harvesting or purifying the antigenfrom a culture containing said antigen but prior to adjuvanting theantigen.
 26. A process for stabilizing an antigen comprising adding hostserum or host albumin to said antigen prior to adjuvanting the antigen.27. A process for stabilizing an antigen comprising adding host sum orhost albumin to said antigen prior to lyophilizing the antigen.
 28. Amethod for eliminating adverse reactions in animals comprisesadministering to said animals an adjuvanted vaccine or an adjuvantedvaccine regimen which is substantially free of non-host albumin.
 29. Amethod for purification of veterinary vaccine antigens comprisingPerfusion chromatography.